CN115299385B - Screening method of golden pomfret backup parent and breeding parent - Google Patents
Screening method of golden pomfret backup parent and breeding parent Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K61/00—Culture of aquatic animals
- A01K61/10—Culture of aquatic animals of fish
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K61/00—Culture of aquatic animals
- A01K61/90—Sorting, grading, counting or marking live aquatic animals, e.g. sex determination
- A01K61/95—Sorting, grading, counting or marking live aquatic animals, e.g. sex determination specially adapted for fish
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K63/00—Receptacles for live fish, e.g. aquaria; Terraria
- A01K63/003—Aquaria; Terraria
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
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- Life Sciences & Earth Sciences (AREA)
- Environmental Sciences (AREA)
- Marine Sciences & Fisheries (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Zoology (AREA)
- Farming Of Fish And Shellfish (AREA)
Abstract
The invention relates to the technical field of aquaculture, in particular to a screening method of a golden pomfret backup parent and a breeding parent. The method obviously improves the sex identification accuracy of the backup parents in the middle period, improves the seed reserving efficiency, can reduce the selection quantity of the backup parents and the breeding parents by 70-80%, improves the breeding production efficiency by 30-40%, greatly reduces the breeding cost and the parent breeding cost, and obviously improves the stress resistance and the growth performance of the breeding offspring.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a screening method of a golden pomfret backup parent and a breeding parent.
Background
Jin Chang is the main cage culture variety in south China, the annual yield reaches 16.8 ten thousand tons, and excellent parents of golden pomfret need to be screened in the artificial breeding, fine breed breeding and scientific experiment processes of golden pomfret. However, male and female golden pomfret have no obvious difference in appearance, and the gonad maturity cannot be identified from the appearance, so that identification of the sex and the gonad maturity of Jin Chang is a difficulty in the artificial breeding process of golden pomfret. Obvious male and female growth difference can occur after the golden pomfret grows to 8 months old, the sex ratio selected and reserved by the backup parent in the early stage cannot be determined, and the culture cost is increased; the gonad maturity cannot be accurately mastered in the parent screening and breeding process, and the ovulation in the same period cannot be achieved, so that the production efficiency is reduced. Therefore, a comprehensive screening method of the golden pomfret backup parent and the breeding parent is sought, the seed reserving efficiency is improved, the selection and reserving proportion of male and female individuals is improved, and a foundation is laid for large-scale real golden pomfret breeding.
Disclosure of Invention
In view of the above, the invention provides a screening method of a golden pomfret backup parent and a breeding parent,
in order to achieve the above object, the present invention provides the following technical solutions:
a screening method of a golden pomfret backup parent and a breeding parent comprises the following steps:
(1) Primary screening of backup parents: selecting 6 months old golden pomfret, selecting 20000 tail with excellent characters as backup parent, and placing into a cement pond for 5 months of cultivation;
(2) Early trait screening: screening early characters of the backup parent groups in the step (1), and placing the backup parent groups in a cement pond for culturing for 1 month;
the early traits include at least one of hypoxia, low temperature, low salt;
(3) Male and female identification and screening: carrying out male and female identification on the parents screened in the step (2) by utilizing ultrasonic and histological detection, recording gender and culturing for 12 months;
(4) Metaphase backup parent screening: selecting golden pomfret with excellent characters from the parents screened in the step (3), wherein the proportion of male and female individuals is 2:1, and placing the golden pomfret in a sea net cage for culturing for 12 months;
(5) Detecting the development degree of male and female gonads: detecting and scoring the sex and the gonadal maturity of the parents screened in the step (4) by utilizing ultrasonic and histological detection;
the scoring indexes comprise the color of male and female gonads, the types, sizes, numbers and structural characteristics of germ cells;
(6) And (3) breeding parent primary screening: selecting golden pomfret with high score and excellent selection characteristics from the step (5) as a propagation parent, wherein the selection and remaining ratio of male and female is 2:1, and culturing for 30-60 days;
(7) Reproductive performance evaluation of the reproductive parents: evaluating the parent reproductive performance screened in the step (6) by utilizing ultrasonic and histological detection, and formulating a high-efficiency grouping reproduction scheme according to an evaluation result;
the reproductive performance indexes comprise: the color of male and female gonads, the type, size, number and structural characteristics of germ cells;
in the step (1), the step (4) and the step (6), the screening criteria for excellent properties include: good growth characteristics, symmetrical body type, good ingestion and quality 20-30% higher than average body quality.
Preferably, in the step (1), the dissolved oxygen of the culture is controlled to be more than 5.0 mg/L.
Preferably, the step (2) specifically includes:
randomly assigning 20000 backup parents of step (1) into 4 groups; a first group of 5000 tails, stressed for 24 hours in a hypoxic environment with a dissolved oxygen of 1.8mg/L, using surviving Jin Chang as a hypoxia tolerant backup parent; a second group of 5000 tails, stressed for 24 hours at a low temperature of 20 ℃, using Jin Chang which survives and is normally ingested as a low temperature resistant backup parent; a third group of 5000 tails, stress for 24 hours in a low-salt environment with salinity below 20%o, taking the surviving Jin Chang as a low-salt-tolerant backup parent; a fourth group of 5000 tails which are used as backup parents for rapid growth of golden pomfret; injecting PIT electronic markers into the four groups of backup parents, placing the four groups of backup parents into a cement pond, and culturing for 1 month for male and female identification and screening.
Preferably, the cement pond for cultivation is 25.0m long, 20.0m wide and 2.5-3.0m deep, and has a cross section of 500-666m 2 The volume is 1250-1500m 3 The bottom of the high water level culture pond is of a funnel-shaped structure, and the gradient of the funnel-shaped structure is not more than 10 degrees.
Preferably, the step (3) specifically includes:
taking 4 groups of parents screened in the step (2), wherein each group comprises at least 1000 parents, carrying out anesthesia by using MS-222, fixing the fishes on an ultrasonic examination table after the anesthesia, and distinguishing the male and female fishes according to different images displayed by the early anatomical features of gonad development of the golden pomfret by using the physical function of ultrasonic waves; and (3) carrying out gonadal tissue trace collection by using a trace tissue collector, placing the collected gonadal tissue on a glass slide for microscopic observation, verifying an ultrasonic identification result, determining male and female sexes, recording corresponding numbers and parent sexes, and placing all marked parents in a sea water net cage for culturing for 12 months.
Preferably, the step (4) specifically includes:
selecting 1200 tails of golden pomfret with good growth character, symmetrical shape, good ingestion and quality 20% -30% higher than average body mass from parents of the step (3), four groups of 300 tails each, recording PIT electronic mark numbers, and placing the female and male individuals in a sea water net cage for culturing for 12 months at a ratio of 2:1.
Preferably, the step (5) specifically includes:
the parent in the step (4) is anesthetized by MS-222, and is fixed on an ultrasonic examination table after anesthesia, and gonadal development degree detection is carried out on different images displayed by the gonadal development characteristics of golden pomfret by utilizing the physical function of ultrasonic waves; and a trace tissue collector is adopted to obtain trace gonad tissue of Jin Chang after anesthesia, the sex and gonad maturity of golden pomfret are detected again according to the color of male and female gonads and the types, sizes, numbers and structural characteristics of germ cells in the male and female gonads, and gonad development maturity scoring is carried out on 4 groups of parents.
In the present invention, female gonadal development maturity scoring criteria are as follows:
1, the method comprises the following steps: the gonads are tightly attached to the body cavity membranes at the two sides below the swim bladder, so that the sex can not be distinguished, and the egg grains can not be seen.
2, the method comprises the following steps: the ovaries are usually in the form of flat bands, and the ovaries are not clearly seen.
3, the method comprises the following steps: the ovaries increase in volume and the ovaries can be seen clearly, but the ovaries cannot be separated and peeled off from the ovary membrane, and the oocytes begin to deposit yolk, but also have earlier oocytes. Egg yolk is not fully filled in egg mass, egg membrane is thickened, and oil globules appear in protoplasm.
4, the following steps: the whole ovary is very large, occupies most of the abdominal cavity, is mostly yellowish or dark yellow, and has very developed connective tissues and blood vessels. Egg grains are filled with yolk, large and full.
5, the method comprises the following steps: the gonads are fully mature, the ovaries are soft, the ovaries are already arranged in the ovaries, the mature ovaries flow out when the abdomen is lightly pressed, and the mature ovaries are transparent.
The scoring criteria for male gonadal development maturity are as follows:
1, the method comprises the following steps: is in a thin line shape and is tightly attached to the body cavity membranes at the two sides under the air bladder. On tissue sections, a dispersed distribution of spermatogonial cells can be seen.
2, the method comprises the following steps: in the form of a thread or ribbon, on a tissue section, it can be seen that the spermatogonia are increased, arranged in bundles, forming solid tubules separated by connective tissue.
3, the method comprises the following steps: is in a round rod shape, and does not flow out semen when extruding the abdomen of the male fish. The lumen appears in the center of the solid fine tube, the tube wall is one to several layers of primary spermatocyte with the same type and the same maturation grade, and the outer surface of the tube wall is surrounded by seminal vesicle cells.
4, the following steps: primary spermatocytes (large volume, deep staining), secondary spermatocytes (smaller volume, deeper staining) and sperm cells (smallest volume, deepest staining) can be seen on the testis section. When few sperm are present in the lumen.
5, the method comprises the following steps: each tubule (in essence, small seminal vesicle) is filled with sperm, and when the abdomen is pressed by lifting, a large amount of thicker milky white semen leaks out of the space Kong Chongchu.
Preferably, the step (6) specifically includes:
according to the gonadal development maturity scoring result of the step (4), 150 golden pomfrets with the score of 3-4, good growth character, well-balanced body type, good ingestion, health and high gonadal development maturity are selected as propagation parents in each group, the total number of the four groups is 600, the selection and the remaining ratio of male and female is 2:1, and the propagation parents are obtained by graded and intensified cultivation for 30-60 days. In the step, the fish with score of 3-4 is selected as the breeding parent according to the scoring result, and the oxytocin usage amount is determined according to the specific scoring, namely, the usage amount of the oxytocin of the parent with low scoring is more than that of the parent with high scoring, so that the effect of the hormone usage amount can be effectively reduced, and meanwhile, the method is also an advantage of continuous parent selection, and meanwhile, the cost is reduced.
Preferably, the step (7) specifically includes:
and (3) anaesthetizing the reproductive parents obtained in the step (6) by utilizing MS-222, fixing the anaesthetized parents on an ultrasonic examination table, checking the gonad development state again by utilizing the physical function of ultrasonic waves, obtaining the micro gonad tissue of Jin Chang after anaesthetization by adopting a micro tissue collector, evaluating the reproductive performance of golden pomfret according to the colors of male and female gonads and the types, sizes, numbers and structural characteristics of germ cells, selecting parents with high egg mass, good sperm quality and gonad development maturity score of 4-5 minutes according to the evaluation result, and formulating a high-efficiency grouping reproductive scheme.
The grouping propagation includes:
grouping the breeding parents, wherein each group comprises 2 female fish tails and 1 male fish tail, and placing the breeding parents in a breeding cage with the length of 5m, the width of 5m and the depth of 4.5m; according to different specific characters, including hypoxia tolerance, low temperature tolerance, low salt tolerance and fast growth, different propagation pairing schemes are formulated, and different groups are placed in a large-scale net cage for separation; artificial spawning is performed aiming at the gonad maturation state of the breeding parents.
In a specific embodiment of the invention, the screening method of the golden pomfret backup parent and the propagation parent comprises the following steps:
(1) Primary screening of backup parents: performing backup parent preliminary screening on 6-month-old golden pomfret seedlings, selecting Jin Chang 20000 tails which are good in growth character, symmetrical in shape, good in ingestion and 20% -30% higher than the average body mass as backup parents, placing the backup parents into a cement pond, temporarily culturing for 5 months, performing early character screening, controlling the dissolved oxygen to be above 5.0mg/L, and temporarily culturing the cement pond to be 25.0m long, 20.0m wide and 2.5-3.0m deep, thereby constructing the cement pond with the cross section of 500-666m 2 The volume is 1250-1500m 3 The bottom of the high water level culture pond is of a funnel-shaped structure, the gradient of the funnel-shaped structure is not more than 10 degrees, and waste discharge and cleaning during culture are facilitated.
(2) Early trait screening: 20000 backup parents were randomly assigned to 4 groups. A first group of 5000 tails, jin Chang which survived stress for 24 hours in a hypoxic environment with a dissolved oxygen of 1.8mg/L, served as a hypoxia tolerant backup parent; a second group of 5000 tails, jin Chang which survive and normally feed under stress for 24 hours at a temperature of 20 ℃ under "low temperature" conditions, served as a low temperature resistant backup parent; a third group of 5000 tails, jin Chang which can survive after being stressed for 24 hours under the low-salt environment with the salinity below 20 per mill, is used as a low-salt-resistant backup parent; and the fourth 5000 tails are used as quick-growth backup parents of golden pomfret. All 4 groups of backup parents (4000 at least 1000) with specific characters are injected with PIT electronic markers and placed in a cement pond for temporary culture for 1 month for male and female identification and screening, the temporary culture cement pond is 25.0m long, 20.0m wide and 2.5-3.0m deep, and the cross section is 500-666m 2 The volume is 1250-1500m 3 The bottom of the high water level culture pond is of a funnel-shaped structure, the gradient of the funnel-shaped structure is not more than 10 degrees, and waste discharge and cleaning during culture are facilitated.
(3) Male and female identification and screening: carrying out female and male identification on 4 groups of backup parents (4000 parents, at least 1000 parents in each group) with different specific characters, firstly carrying out anesthesia by utilizing MS-222, fixing the fish on an ultrasonic examination table after anesthesia, and distinguishing the female and male of different images displayed by anatomical features of early gonad development of golden pomfret by utilizing the physical function of ultrasonic waves; and (3) carrying out gonadal tissue trace collection by using a trace tissue collector, placing the collected gonadal tissue on a glass slide for microscopic observation, verifying an ultrasonic identification result, determining sexes of male and female, recording corresponding PIT numbers and the sexes of backup parents, and screening the middle-stage backup parents after all marked backup parents are placed in a seawater net cage for 12 months.
(4) Metaphase backup parent screening: among the 4 groups of backup parents (1000 tails) with specific characters, 300 tails of golden pomfret with good growth characters, symmetrical body type, good ingestion, health and quality 20% -30% higher than the average body quality are selected as the middle-stage backup parents, PIT electronic mark numbers are recorded, the proportion of male and female individuals is selected to be 2:1, and all the middle-stage backup parents (1200 tails, 300 tails in each group) are placed in a sea water net cage for cultivation for 12 months, and then the male and female gonad development degree is detected.
(5) Detecting the development degree of male and female gonads: detecting male and female gonad development degrees of 4 groups of intermediate backup parents (1200 tails and 300 tails in each group) with different specific characters, firstly, anesthetizing by using MS-222, and detecting gonad development degrees of different images displayed by gonad development characteristics of golden pomfret by using physical functions of ultrasonic waves after anesthesia; and a trace tissue collector is adopted to obtain a trace gonadal tissue of Jin Chang after anesthesia, the sex and gonadal maturity of golden pomfret are detected again according to the color of male and female gonads and the types, sizes, numbers and structural characteristics of germ cells in the male and female gonads, the gonadal development maturity of 4 groups of metaphase backup parents with different specific characters is scored, and then the primary screening of the breeding parents is carried out.
(6) And (3) breeding parent primary screening: aiming at the gonad development maturity scoring result of the intermediate backup parents, 150 (total 600) gonad development maturity scoring is high (3 and 4 minutes), golden pomfret with good growth characters, symmetrical body type, good ingestion and high gonad development maturity is selected as a propagation parent, the female and male selection and remaining ratio is 2:1, a targeted enhanced cultivation scheme is designed, the content of feeding proteins is properly increased (3-minute backup parents) or reduced (4-minute backup parents), blindness of parent cultivation and parent cultivation cost are reduced, and propagation parent reproduction performance evaluation is carried out after grading enhanced cultivation is carried out for 30-60 days before all selected propagation parents are propagated. .
(7) Reproductive performance evaluation of the reproductive parents: the reproduction performance of the reproduction parents (600 tails, 150 tails in each group) of 4 groups with different specific characters is evaluated, PIT electronic marking is matched after the reproduction parents of golden pomfret are subjected to intensified cultivation, MS-222 is firstly utilized for anesthesia, the physical function of ultrasonic waves is utilized for checking the gonad development state again after anesthesia, a trace gonad tissue of Jin Chang after anesthesia is obtained by a trace tissue collector, the reproduction performance (spawning quantity, sperm quality and the like) of golden pomfret is evaluated according to the colors of male and female gonads and the types, sizes, numbers and structural characteristics of germ cells in the female and male gonads, and a high-efficiency grouping reproduction scheme is formulated according to the evaluation result, wherein the parents with high spawning quantity, good sperm quality and high gonad development maturity score (4 and 5 minutes) are selected. .
(8) And (3) making a grouping propagation scheme: for 4 groups of propagation parents (600 tails, 150 tails in each group) with different specific characters, the proportion of male to female is 2:1, a grouping propagation scheme is formulated, different propagation pairing schemes can be formulated according to different specific characters including hypoxia tolerance, low temperature tolerance, low salt tolerance and fast growth, an artificial induced spawning scheme is designed aiming at the gonad maturation state of the propagation parents, the hormone usage amount is reduced, the cost is reduced, and the induced spawning efficiency is improved.
The male individual has a diaphragm outside the gonad, has low fat content, and shows bright echo images with fine textures under ultrasonic detection. From stage II, the testis tissue shows a highly reflective structure, shows a smooth, narrow, surface wrinkled character, with sharp edges, the image shows a distinctly smooth area and the edges are curved or there are several areas of rounded edges. Stage III and stage IV testis tissues under ultrasonic waves show that surface wrinkles are obviously increased, sharp edges are gradually smoothed, gonad width is increased, and dense texture bright areas of echo images are increased.
The gonad of female individual has no external diaphragm, is wrapped by fat, and shows more complex echo images under ultrasonic detection. The first-stage ovarian tissue is positioned on the lateral surface of the gonad, the gonad is mainly composed of anechoic fat, the second-stage ovarian tissue is arranged in a hyperechoic area, but the second-stage ovarian tissue is not obvious, and the gonad is mainly composed of anechoic fat; ovaries stage III and IV, single eggs with granular contents are visible in the echogram. Oocytes are different in size and in black background from adipose tissue. Ovarian tissue has a granular and heterogeneous structure.
Because of the limitations of the super technology, the gonad identification of the parent fish by adopting the method has higher gonad development requirements on the parent fish, the gonad of the female fish is required to be at a level above the III phase, and the gonad of the male fish is required to be at a level above the III phase. In order to eliminate the influence of excessive fat of gonads of parent fishes on super-examination as much as possible, the parent fishes need to be starved for about one month so as to reduce the fat content of the gonads of the parent fishes.
The ultrasonic detection has the advantages of reducing fish injury, but has lower detection accuracy, needs to be matched with a trace tissue collector to conduct trace collection of gonadal tissues, and places the collected gonadal tissues on a glass slide to conduct microscopic observation on the types, sizes, numbers and structural characteristics of germ cells to determine sex, gonadal development state and reproductive performance evaluation.
(9) The seed reserving proportion of the invention can be adjusted according to specific conditions.
(10) The invention carries out continuous simultaneous screening for multiple times on character selection and sex selection, and improves seed reserving and breeding efficiency.
The screening method provided by the invention obviously improves the sex identification accuracy of the backup parent in the middle period, improves the seed reserving efficiency, reduces the selection quantity of the backup parent and the breeding parent by 70-80%, improves the breeding production efficiency by 30-40%, and improves the stress resistance and the growth performance of the breeding offspring.
Drawings
FIG. 1 shows a technical roadmap of the screening method of the invention;
FIG. 2 shows the results of slice identification of male and female sex in early gonadogenesis of golden pomfret, wherein 2-A is female and 2-B is male;
FIG. 3 shows the results of detection of male and female gonadal development degree of golden pomfret, wherein 3-A is female and 3-B is male;
FIG. 4 shows evaluation of reproductive performance of golden pomfret reproductive parents wherein 4-A is female and 4-B is male;
FIG. 5 shows backup parent selection;
FIG. 6 shows reproductive parental usage;
FIG. 7 is a graph showing a comparison of the use of oxytocin and spawning parents used with the same number of fertilized eggs;
figure 8 shows the fertilization rate, hatchability and survival rate comparisons for the experimental and control groups.
Detailed Description
The invention provides a screening method of a golden pomfret backup parent and a breeding parent. Those skilled in the art can, with the benefit of this disclosure, suitably modify the process parameters to achieve this. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the invention.
The test materials adopted by the invention are all common commercial products and can be purchased in the market.
The invention is further illustrated by the following examples:
example 1
(1) Primary screening of backup parents: the 6 month old golden pomfret seedlings are subjected to backup parent primary screening, jin Chang 20000 tails with good growth characteristics, uniform body shape, good ingestion and quality 20% -30% higher than the average body quality are selected as backup parents, and are placed in a cement pond for temporary culture for 5 months, and then early-stage character screening is performed.
(2) Early trait screening: 20000 backup parents were randomly assigned to 4 groups. A first group of 5000 tails, jin Chang which survived stress for 24 hours in a hypoxic environment with a dissolved oxygen of 1.8mg/L, served as a hypoxia tolerant backup parent; a second group of 5000 tails, jin Chang which survive and normally feed under stress for 24 hours at a temperature of 20 ℃ under "low temperature" conditions, served as a low temperature resistant backup parent; a third group of 5000 tails, jin Chang which can survive after being stressed for 24 hours under the low-salt environment with the salinity below 20 per mill, is used as a low-salt-resistant backup parent; and the fourth 5000 tails are used as quick-growth backup parents of golden pomfret. All 4 groups of backup parents (4000 at least 1000 parents each) with specific traits are placed in a cement pond for temporary rearing for 1 month for male and female identification and screening.
(3) Male and female identification and screening: carrying out female and male identification on 4 groups of backup parents (4000 parents, at least 1000 parents in each group) with different specific characters, firstly carrying out anesthesia by utilizing MS-222, and distinguishing female and male images according to different images displayed by the early anatomical features of gonad development of golden pomfret by utilizing the physical function of ultrasonic waves after anesthesia; and (3) carrying out gonadal tissue trace collection by using a trace tissue collector, placing the collected gonadal tissue on a glass slide for microscopic observation, verifying an ultrasonic identification result, determining male and female sexes (figure 2), injecting PIT electronic marks, recording corresponding numbers and the sex of backup parents, and screening the middle-stage backup parents after all marked backup parents are placed in a seawater net cage for 12 months.
(4) Metaphase backup parent screening: among the 4 groups of backup parents (1000 tails) with specific characters, 300 tails of golden pomfret with good growth characters, symmetrical body type, good ingestion, health and quality 20% -30% higher than the average body quality are selected as the middle-stage backup parents, PIT electronic mark numbers are recorded, the proportion of male and female individuals is selected to be 2:1, and all the middle-stage backup parents (1200 tails, 300 tails in each group) are placed in a sea water net cage for cultivation for 12 months, and then the male and female gonad development degree is detected.
(5) Detecting the development degree of male and female gonads: detecting male and female gonad development degrees of 4 groups of intermediate backup parents (1200 tails and 300 tails in each group) with different specific characters, firstly, anesthetizing by using MS-222, and detecting gonad development degrees of different images displayed by gonad development characteristics of golden pomfret by using physical functions of ultrasonic waves after anesthesia; and obtaining the trace gonadal tissue of Jin Chang after anesthesia by adopting a trace tissue collector, detecting the sex and gonadal maturity of golden pomfret again according to the color of male and female gonads and the types, sizes, numbers and structural characteristics of germ cells in the male and female gonads (figure 3), scoring the gonadal development maturity of the intermediate backup parents of 4 groups with different specific characters, and then performing primary screening of the breeding parents.
In the present invention, female gonadal development maturity scoring criteria are as follows:
1, the method comprises the following steps: the gonads are tightly attached to the body cavity membranes at the two sides below the swim bladder, so that the sex can not be distinguished, and the egg grains can not be seen.
2, the method comprises the following steps: the ovaries are usually in the form of flat bands, and the ovaries are not clearly seen.
3, the method comprises the following steps: the ovaries increase in volume and the ovaries can be seen clearly, but the ovaries cannot be separated and peeled off from the ovary membrane, and the oocytes begin to deposit yolk, but also have earlier oocytes. Egg yolk is not fully filled in egg mass, egg membrane is thickened, and oil globules appear in protoplasm.
4, the following steps: the whole ovary is very large, occupies most of the abdominal cavity, is mostly yellowish or dark yellow, and has very developed connective tissues and blood vessels. Egg grains are filled with yolk, large and full.
5, the method comprises the following steps: the gonads are fully mature, the ovaries are soft, the ovaries are already arranged in the ovaries, the mature ovaries flow out when the abdomen is lightly pressed, and the mature ovaries are transparent.
The scoring criteria for male gonadal development maturity are as follows:
1, the method comprises the following steps: is in a thin line shape and is tightly attached to the body cavity membranes at the two sides under the air bladder. On tissue sections, a dispersed distribution of spermatogonial cells can be seen.
2, the method comprises the following steps: in the form of a thread or ribbon, on a tissue section, it can be seen that the spermatogonia are increased, arranged in bundles, forming solid tubules separated by connective tissue.
3, the method comprises the following steps: is in a round rod shape, and does not flow out semen when extruding the abdomen of the male fish. The lumen appears in the center of the solid fine tube, the tube wall is one to several layers of primary spermatocyte with the same type and the same maturation grade, and the outer surface of the tube wall is surrounded by seminal vesicle cells.
4, the following steps: primary spermatocytes (large volume, deep staining), secondary spermatocytes (smaller volume, deeper staining) and sperm cells (smallest volume, deepest staining) can be seen on the testis section. When few sperm are present in the lumen.
5, the method comprises the following steps: each tubule (in essence, small seminal vesicle) is filled with sperm, and when the abdomen is pressed by lifting, a large amount of thicker milky white semen leaks out of the space Kong Chongchu.
(6) And (3) breeding parent primary screening: aiming at the gonad development maturity scoring result of the intermediate backup parent, 150 golden pomfrets (600 total golden pomfrets) with good growth characters, symmetrical body types, good ingestion, health and high gonad development maturity are selected as propagation parents (the male and female ratio is 2:1) in each group, a targeted enhanced cultivation scheme is designed, and the propagation performance of the propagation parents is evaluated after the grading enhanced cultivation of all selected propagation parents for 30-60 days before propagation.
(7) Reproductive performance evaluation of the reproductive parents: the reproduction performance of the reproduction parents (600 tails, 150 tails in each group) of 4 groups with different specific characters is evaluated, after the reproduction parents of golden pomfret are subjected to intensified cultivation, PIT electronic marking is matched, firstly, the MS-222 is used for anesthesia, the physical function of ultrasonic waves is used for checking the gonad development state again after anesthesia, a trace gonad tissue of Jin Chang after anesthesia is obtained by a trace tissue collector, the reproduction performance (spawning quantity, sperm quality and the like) of the golden pomfret is evaluated according to the colors of male and female gonads and the types, sizes, numbers and structural characteristics of germ cells in the male and female gonads (figure 4), and a high-efficiency grouping reproduction scheme is formulated according to the evaluation result.
(8) And (3) making a grouping propagation scheme: for 4 groups of propagation parents (600 tails, 150 tails in each group) with different specific characters, the proportion of male to female is 2:1, a grouping propagation scheme is formulated, different propagation pairing schemes can be formulated according to different specific characters including hypoxia tolerance, low temperature tolerance, low salt tolerance and fast growth, and an artificial induced spawning scheme is designed aiming at the gonad maturation state of the propagation parents, so that the induced spawning efficiency is improved.
Example 2
Experiments are carried out on a national standard golden pomfret cultivation teaching practice base of Hainan university, the experiment group is carried out according to the specific steps related by the invention, and the comparison is carried out by a cooperation enterprise in the existing group selection and group reproduction mode (selection is not performed to distinguish between male and female). The specific process is as follows:
TABLE 1
Table 2 control screening procedure
The results are shown in FIGS. 5 to 8.
From the results, it can be seen that:
1. the selection quantity of the primary screening backup parent of the experimental group is reduced by 80.00% compared with that of the primary screening backup parent of the control group;
2. the use amount of the experimental group propagation parents is reduced by 70.00% compared with that of the control group propagation parents; the breeding and cultivating cost of the experimental group breeding parents is reduced by 60-70% compared with that of the control group breeding parents;
3. the participation rate of the experimental group propagation parents is increased by 54.68% compared with that of the control group propagation parents;
4. the utilization rate of the oxytocin in the experimental group is reduced by 40.00% compared with that of the oxytocin in the control group;
5. the fertilization rate, the hatching rate and the survival rate of the experimental group are respectively improved by 8.07 percent, 5.41 percent and 29.84 percent compared with the control group;
6. the comprehensive propagation efficiency is improved by 42.26 percent.
Claims (9)
1. The screening method of the golden pomfret backup parent and the breeding parent is characterized by comprising the following steps:
(1) Primary screening of backup parents: selecting 6 months old golden pomfret, selecting 20000 tail with excellent characters as backup parent, and placing into a cement pond for 5 months of cultivation;
(2) Early trait screening: screening early characters of the backup parent groups in the step (1), and placing the backup parent groups in a cement pond for culturing for 1 month;
the early traits include at least one of hypoxia, low temperature, low salt;
(3) Male and female identification and screening: carrying out male and female identification on the parents screened in the step (2) by utilizing ultrasonic and histological detection, recording gender and culturing for 12 months;
(4) Metaphase backup parent screening: selecting golden pomfret with excellent characters from the parents screened in the step (3), wherein the proportion of male and female individuals is 2:1, and placing the golden pomfret in a sea net cage for culturing for 12 months;
(5) Detecting the development degree of male and female gonads: detecting and scoring the sex and the gonadal maturity of the parents screened in the step (4) by utilizing ultrasonic and histological detection;
the scoring indexes comprise the color of male and female gonads, the types, sizes, numbers and structural characteristics of germ cells;
(6) And (3) breeding parent primary screening: selecting golden pomfret with high score and excellent selection characteristics from the step (5) as a propagation parent, wherein the selection and remaining ratio of male and female is 2:1, and culturing for 30-60 days;
(7) Reproductive performance evaluation of the reproductive parents: evaluating the parent reproductive performance screened in the step (6) by utilizing ultrasonic and histological detection, and formulating a high-efficiency grouping reproduction scheme according to an evaluation result;
the reproductive performance indexes comprise: the color of male and female gonads, the type, size, number and structural characteristics of germ cells;
in the step (1), the step (4) and the step (6), the screening criteria for excellent properties include: good growth character, symmetrical body shape, good ingestion and quality 20-30% higher than average body quality;
the step (2) specifically comprises:
randomly assigning 20000 backup parents of step (1) into 4 groups; a first group of 5000 tails, stressed for 24 hours in a hypoxic environment with a dissolved oxygen of 1.8mg/L, using surviving Jin Chang as a hypoxia tolerant backup parent; a second group of 5000 tails, stressed for 24 hours at a low temperature of 20 ℃, using Jin Chang which survives and is normally ingested as a low temperature resistant backup parent; a third group of 5000 tails, stress for 24 hours in a low-salt environment with salinity below 20%o, taking the surviving Jin Chang as a low-salt-tolerant backup parent; a fourth group of 5000 tails which are used as backup parents for rapid growth of golden pomfret; injecting PIT electronic markers into the four groups of backup parents, placing the four groups of backup parents into a cement pond, and culturing for 1 month for male and female identification and screening.
2. The method according to claim 1, wherein in the step (1), dissolved oxygen in the culture is controlled to be 5.0mg/L or more.
3. The screening method according to claim 1, wherein the cement pond for cultivation is 25.0m long, 20.0m wide and 2.5-3.0m deep, and has a cross section of 500-666m 2 The volume is 1250-1500m 3 The bottom of the high water level culture pond is of a funnel-shaped structure, and the gradient of the funnel-shaped structure is not more than 10 degrees.
4. The screening method according to claim 1, wherein step (3) specifically comprises:
taking 4 groups of parents screened in the step (2), wherein each group comprises at least 1000 parents, carrying out anesthesia by using MS-222, fixing the fishes on an ultrasonic examination table after the anesthesia, and distinguishing the male and female fishes according to different images displayed by the early anatomical features of gonad development of the golden pomfret by using the physical function of ultrasonic waves; and (3) carrying out gonadal tissue trace collection by using a trace tissue collector, placing the collected gonadal tissue on a glass slide for microscopic observation, verifying an ultrasonic identification result, determining male and female sexes, recording corresponding numbers and parent sexes, and placing all marked parents in a sea water net cage for culturing for 12 months.
5. The screening method according to claim 1, wherein the step (4) specifically comprises:
selecting 1200 tails of golden pomfret with good growth character, symmetrical shape, good ingestion and quality 20% -30% higher than average body mass from parents of the step (3), four groups of 300 tails each, recording PIT electronic mark numbers, and placing the female and male individuals in a sea water net cage for culturing for 12 months at a ratio of 2:1.
6. The screening method according to claim 1, wherein the step (5) specifically comprises:
the parent in the step (4) is anesthetized by MS-222, and is fixed on an ultrasonic examination table after anesthesia, and gonadal development degree detection is carried out on different images displayed by the gonadal development characteristics of golden pomfret by utilizing the physical function of ultrasonic waves; and a trace tissue collector is adopted to obtain trace gonad tissue of Jin Chang after anesthesia, the sex and gonad maturity of golden pomfret are detected again according to the color of male and female gonads and the types, sizes, numbers and structural characteristics of germ cells in the male and female gonads, and gonad development maturity scoring is carried out on 4 groups of parents.
7. The screening method according to claim 1, wherein the step (6) specifically comprises:
according to the gonadal development maturity scoring result of the step (4), 150 golden pomfrets with the score of 3-4, good growth character, well-balanced body type, good ingestion, health and high gonadal development maturity are selected as propagation parents in each group, the total number of the four groups is 600, the selection and the remaining ratio of male and female is 2:1, and the propagation parents are obtained by graded and intensified cultivation for 30-60 days.
8. The screening method according to claim 1, wherein the step (7) specifically comprises:
and (3) anaesthetizing the reproductive parents obtained in the step (6) by utilizing MS-222, fixing the anaesthetized parents on an ultrasonic inspection table, checking the gonad development state again by utilizing the physical function of ultrasonic waves, obtaining the micro gonad tissue of Jin Chang after anaesthetization by adopting a micro tissue collector, evaluating the reproductive performance of golden pomfret according to the colors of male and female gonads and the types, sizes, numbers and structural characteristics of germ cells, selecting parents with high egg mass, good sperm quality and gonad development maturity score of 4-5 according to the evaluation result, and making a high-efficiency grouping reproductive scheme for grouping reproduction.
9. The screening method according to any one of claims 1 to 8, wherein the packet reproduction comprises:
grouping the breeding parents, wherein each group comprises 2 female fish tails and 1 male fish tail, and placing the breeding parents in a breeding cage with the length of 5m, the width of 5m and the depth of 4.5m; according to different specific characters, including hypoxia tolerance, low temperature tolerance, low salt tolerance and fast growth, different propagation pairing schemes are formulated, and different groups are placed in a large-scale net cage for separation; artificial spawning is performed aiming at the gonad maturation state of the breeding parents.
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| CN101766495A (en) * | 2008-12-29 | 2010-07-07 | 中国水产科学研究院东海水产研究所 | Method for identifying sex of sturgeon by ultrasonic waves |
| CN109819914A (en) * | 2019-03-28 | 2019-05-31 | 海南晨海水产有限公司 | A kind of artificial hybridization breeding method of egg-shaped pompano and Bu Shi silvery pomfret Scad |
| CN111869604A (en) * | 2020-07-17 | 2020-11-03 | 广西壮族自治区水产科学研究院 | Construction method of egg-shaped pompano full-sib family |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101766495A (en) * | 2008-12-29 | 2010-07-07 | 中国水产科学研究院东海水产研究所 | Method for identifying sex of sturgeon by ultrasonic waves |
| CN109819914A (en) * | 2019-03-28 | 2019-05-31 | 海南晨海水产有限公司 | A kind of artificial hybridization breeding method of egg-shaped pompano and Bu Shi silvery pomfret Scad |
| CN111869604A (en) * | 2020-07-17 | 2020-11-03 | 广西壮族自治区水产科学研究院 | Construction method of egg-shaped pompano full-sib family |
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